PROJECT SUMMARY Mosquitoes from the Cx. pipiens complex are principal vectors of lymphatic filariasis worms and encephalitis viruses, including West Nile virus, which is now the most common arboviral disease in the USA. To facilitate the development of advanced genome-based strategies for vector control, high-quality genome assemblies, based on third-generation sequencing technologies, were developed for species of the Anopheles gambiae complex, as well as for Aedes aegypti and Aedes albopictus. However, the genome assembly for Cx. pipiens, the type species in the complex, is still missing. Our project will develop high-quality genome assemblies for two members of the complex, Cx. p. pipiens and Cx. p. molestus. Ecological, behavioral, and physiological differences between closely related taxa are often associated with chromosomal inversions. However, very few chromosomal inversions have been described in Culex mosquitoes due to the inferior quality of their polytene chromosomes. Our project will test whether any of the chromosomal rearrangements are taxa-specific or related to local adaptation of Cx. pipiens mosquitoes to the environment. Specific aims for this project are to: 1) develop high-quality genome assemblies for Cx. p. pipiens and Cx. p. molestus; 2) create physical maps of the genomes of Cx. p. pipiens and Cx. p. molestus; and 3) identify chromosomal rearrangements in Cx. p. pipiens and Cx. p. molestus. We will utilize the cutting-edge genomic technologies of Oxford Nanopore Technology sequencing, BioNano optical mapping, Hi-C scaffolding, and cDNA-based physical mapping. The Hi-C approach and fluorescence in situ hybridization will be used to identify chromosomal inversions in Cx. p. pipiens and Cx. p. molestus. We envision that development of high- quality genome assemblies for two members of the Cx. pipiens complex, and identification of their chromosomal rearrangements, will further stimulate the development of new strategies for vector control.